Heparan sulfates (HSs) are highly sulfated polysaccharides, present on the surface of mammalian cells and in the extracellular matrix in large quantities. HSs play critical roles in a variety of important biological processes, including assisting viral infection, regulating blood coagulation and embryonic development, suppressing tumor growth, and controlling the eating behavior of mice by interacting with specific regulatory proteins (Liu, J., and Thorp, S. C. (2002) Med. Res. Rev. 22:1-25; Rosenberg, R. D., et al., (1997) J. Clin. Invest. 99:2062-2070; Bernfield, M., et al., (1999) Annu. Rev. Biochem. 68:729-777; Alexander, C. M., et al., (2000) Nat. Genet 25:329-332; Reizes, O., et al., (2001) Cell 106:105-116). HS polysaccharides carry negative charges under physiological pH, and the disaccharide repeating units include 1→4-linked sulfated glucosamine and uronic acid. The unique sequences determine to which specific proteins HSs bind, thereby regulating biological processes.
The biosynthesis of HS occurs in the Golgi apparatus. It is initially synthesized as a copolymer of glucuronic acid and N-acetylated glucosamine by D-glucuronyl and N-acetyl-D-glucosaminyltransferase, followed by various modifications (Lindahl, U., et al., (1998) J. Biol. Chem. 273:24979-24982).
These modifications include N-deacetylation and N-sulfation of glucosamine, C5 epimerization of glucuronic acid to form iduronic acid residues, 2-O-sulfation of iduronic and glucuronic acid, as well as 6-O-sulfation and 3-O-sulfation of glucosamine. Several enzymes that are responsible for the biosynthesis of HS have been cloned and characterized (Esko, J. D., and Lindahl, U. (2001) J. Clin. Invest. 108:169-173). These enzymes have become essential tools for investigating the relationship between the structures and functions of HS.
What is still unknown is the detailed mechanism for regulating the biosynthesis of HS with a defined saccharide sequence. A recent report (Liu, J., et al., (1999) J. Biol. Chem. 274:5185-5192) suggests that the expression levels of various HS biosynthetic enzyme isoforms contribute to the synthesis of specific saccharide sequences in specific tissues. HS N-deacetylase/N-sulfotransferase, 3-O-sulfotransferase, and 6-O-sulfotransferase are present in multiple isoforms. Each isoform is believed to recognize a saccharide sequence around the modification site in order to generate a specific sulfated saccharide sequence (Liu, J., et al., (1999) J. Biol. Chem. 274:5185-5192 ; Aikawa, J.-I., et al., (2001) J. Biol. Chem. 276:5876-5882; Habuchi, H., et al., (2000) J. Biol. Chem. 275:2859-2868). For instance, HS D-glucosaminyl3-O-sulfotransferase (3-OST) isoforms generate 3-O-sulfated glucosamine residues that are linked to different sulfated uronic acid residues. 3-OST isoform 1 (3-OST-1) transfers sulfate to the 3-OH position of an N-sulfated glucosamine residue that is linked to a glucuronic acid residue at the nonreducing end (GlcUA—GlcNS±6S). However, 3-OST isoform 3 (3-OST-3) transfers sulfate to the 3-OH position of an N-unsubstituted glucosamine residue that is linked to a 2-O-sulfated iduronic acid at the nonreducing end (IdoUA2S—GlcNH2±6S) (Liu, J., et al., (1999) J. Biol. Chem. 274:38155-38162). The difference in the substrate specificity of 3-OSTs results in distinct biological functions. For example, the HS modified by 3-OST-1 binds to antithrombin (AT) and possesses anticoagulant activity (Liu, J., et al., (1996) J. Biol. Chem. 271:27072-27082). However, the HS modified by 3-OST-3 (3-OST-3A and 3-OST-3B) binds to glycoprotein D (gD) of herpes simplex virus, type 1, (HSV-1) thus mediating viral entry (Shukla, D., et al., (1999) Cell 99:13-22).
The HS—and heparin-regulated anticoagulation mechanisms have been studied extensively. It is now known that HS and heparin interact with AT, a serine protease inhibitor, to inhibit the activities of thrombin and factor Xa in the blood coagulation cascade (Rosenberg, R. D., et al., (1997) J. Clin. Invest. 99:2062-2070). Anticoagulant-active HS (HSact) and heparin contain one or multiple AT-binding sites per polysaccharide chain. This binding site contains a specific pentasaccharide sequence with a structure of —GlcNS(or Ac)6S—GlcUA—GlcNS3S(±6S)—IdoUA2S—GlcNS6S—. The 3-O-sulfation of glucosamine for generating GlcNS3S(±6S) residue, which is carried out by 3-OST-1 (EC 2.8.2.23), is the critical modification for the synthesis of HSact (Liu, J., et al., (1996) J. Biol. Chem. 271:27072-27082; Shworak, N. W. , et al., (1997) J. Biol. Chem. 272:28008-28019).
Cell surface HS also assists HSV-1 infection (WuDunn, D., and Spear, P. G. (1989) J. Virol. 63:52-58). A recent report (Shukla, D., et al., (1999) Cell 99:13-22) suggests that a specific 3-O-sulfated HS is involved in assisting HSV-1 entry. The 3-O-sulfated HS is generated by 3-OST-3 but not by 3-OST-1. In addition, the 3-O-sulfated HS provides binding sites for HSV-1 envelope glycoprotein D, which is a key viral protein involved in the entry of HSV-1 (Shukla, D., et al., (1999) Cell 99:13-22). Because 3-OST-3-modified HS is rarely found in HS from natural sources, the study suggests that HSV-1 recognizes a unique saccharide structure. Indeed, the result from the structural characterization of a gD-binding octasaccharide revealed that the octasaccharide possesses a specific saccharide sequence (Liu, J., et al., (2002) J. Biol. Chem. 277:33456-33467). In addition, the binding affinity of the 3-O-sulfated HS for gD is about 2 μM (Shukla, D., et al., Cell 99:13-22). This affinity is similar to that reported for the binding of gD to the protein receptors, suggesting that HSV-1 utilizes both protein and HS cell surface receptors to infect target cells (Willis, S. H., et al., (1998) J. Virol. 72:5938-5947; Krummenacher, C., et al., (1999) J. Virol. 73:8127-8137). It is believed that the interaction between gD and the 3-O-sulfated HS or the protein entry receptors somehow triggers the fusion between the virus and the cell in the presence of other viral envelope proteins, including gB, gH, and gL (Shukla, D., and Spear, P. G. (2001) J. Clin. Invest. 108:503-510). A study of the co-crystal structure of gD and herpes entry receptor HveA suggests that the binding of HveA to gD induces conformational changes in gD (Carfi, A., et al., (2001) Mol. Cell 8:169-179).
Therefore, a need persists for additional tools for both understanding the mechanism for the biosynthesis of the biologically active HS and for investigating the relationship between the saccharide sequences and the biological functions of HS. This and other needs are addressed by the present disclosure.